Northern blot analysis of total RNA prepared from immature (27-day-old) rat ovaries revealed the presence of a single p53 messenger RNA (mRNA) transcript (2.0 kilobases) and multiple WT-1 messages (1.8, 3.5, and 7.5 kilobases). Subcutaneous injection of immature rats with 10 IU equine CG (eCG) reduced the levels of p53 and WT-1 mRNA to 71 +/- 9% (P < 0.05) and 46 +/- 3% (P < 0.05), respectively, of saline-treated control levels after 2 days. The inhibition of tumor suppressor gene expression by eCG treatment was associated with a marked reduction in the number of apoptotic granulosa cells and atretic follicles. Furthermore, immunohistochemical analysis revealed that p53 protein was localized exclusively to nuclei of apoptotic granulosa cells of atretic follicles, and that p53 immunostaining was reduced to undetectable levels after in vivo treatment with eCG.

To further evaluate whether granulosa cell apoptosis is linked to increased expression of tumor suppressor genes, we analyzed levels of p53 and WT-1 mRNA in antral follicles induced to undergo atresia in vitro by serum-free culture. Healthy antral follicles snap-frozen immediately after isolation from eCG-primed rat ovaries (0-h, no apoptosis) possessed relatively low, but detectable, levels of p53 and WT-1 mRNA; however, p53 mRNA levels were increased to 147 +/- 3% (P < 0.05) of 0 h control levels after a 24-h incubation without trophic hormone support (extensive apoptosis). In contrast, WT-1 mRNA levels remained constitutive in follicles incubated for 24 h compared to follicles snap-frozen immediately after isolation. We conclude that the p53 and WT-1 genes are expressed and gonadotropin regulated in the ovary and propose that the products of these genes serve as important regulators of granulosa cell fate during follicle development.